Validação do miR-17-5p e miR-637 na expressão e regulação dos genes VEGFA e NFE2L2 em câncer de cabeça e pescoço in vitro

Abstract

Head and Neck Cancer (HNC) includes tumors located on tongue, oral cavity, larynx, oropharynx, hypopharynx, pharynx and paranasal sinuses. Angiogenesis is the formation of new blood vessels from pre-existing vessels, leading to nutrition and tumor development. Vascular Endothelial Growth Factor (VEGF) is fundamental to angiogenic process. Nuclear Erythroid Factor 2-like2 (NFE2L2) is the main regulator of cellular oxidative stress. MicroRNAs (miRNAs) are small RNA molecules consisting of approximately 22 nucleotides involved in gene regulation. Objectives: To evaluate gene and protein expression of VEGFA and NFE2L2 and expression of hsa-miR-17-5p and hsa-miR-637 miRNAs in HNC cell lines; to investigate the relationship between genes and micros. Materials and Methods: HEp-2, FaDu and HN13 cell lines were used. Quantifications of genes and miRNAs were performed using quantitative real-time PCR (qPCR) technique. Quantification of VEGFA and NFE2L2 proteins was performed by Western Blotting. To evaluate gene regulation by miRNAs, the lines were transfected with miR-17-5p inhibitor and miR-637 mimic; expression of VEGFA and NFE2L2 genes was quantified. Results: VEGFA was not regulated by miR-637 in HEp-2 and HN13 lines. In FaDu, there was an increase in VEGFA expression (p = 0.0313). In relation to miR-17-5p, VEGFA had its expression increased in HEp-2 (p = 0.1563) and HN13 (p = 0.1563) lines, and there was no regulation of this gene in FaDu (p = 0, 6875). NFE2L2 expression was increased in HEp-2, in cells transfected with miR-637 (p = 0.0313) and with miR-17-5p (p = 0.1563). In FaDu, NFE2L2 expression was also increased with miR-637 (p = 0.0313), but it did not show regulation by miR-17-5p (p = 1.0000). In HN13 there was no regulation in cells with miR-637 (p = 0.8438); with miR-17-5p (p = 0.1250) there was an increase in NFE2L2 expression. In HEp-2 and HN13 lines, VEGFA protein expression was slightly increased after transfection with miR-637 (HEp-2 p = 0.3374; HN13 p = 0.4469), and reduced with miR-17-5p ( HEp-2 p = 0.1948; HN13 p = 0.48047). In FaDu, VEGFA was reduced with miR-637 (p = 0.3414), and increased with miR-17-5p (p = 0.5004). Protein expression values were compared with their respective negative mimics and inhibitor controls. NFE2L2 protein was increased in FaDu and HN13 (p = 0.2792; p = 0.2382) in cells with miR-637 when compared to control (p = 0.1870; p = 0.0393), while in HEp-2 (p = 0.2186) there was a small reduction compared to control (p = 0.2940). In cells transfected with miR-17-5p, in HEp-2 and HN13 lines, there was no difference in expression between transfected cells (p = 0.1714; p = 0.0803) and control (p = 0.1026; p = 0.0743), while in FaDu there was a small reduction (p = 0.2972) compared to control (p = 0.3642). Conclusion: MiR-17-5p is able to regulate expression of VEGFA and NFE2L2 in HNC lines. The results show that there are mechanisms involved in regulation of VEGFA and NFE2L2 genes by hsa-miR-637 and hsa-miR-17-5p miRNAs, however different anatomical sites can influence regulation and expression profile.